Previously, viable chimeric flaviviruses were constructed that contained tick-borne encephalitis virus (TBEV) CME or ME structural protein genes with the remaining genes derived from dengue type 4 virus (DEN4). The ME chimera retained the neurovirulence for mice of its TBEV parent from which its M and E genes were derived, but it lacked the peripheral invasiveness of TBEV. Later, neurovirulence demonstrable by intracerebral (IC) inoculation was reduced or ablated by a single mutation introduced into the M, E or NS1 of the chimera. These mutations also caused a restriction in replication in simian and mosquito cells. Nonetheless, parenteral inoculation of these attenuated mutants induced complete resistance in mice to fatal encephalitis caused by the neurovirulent ME chimera. This suggests a new strategy for developing a live attenuated TBEV vaccine. Langat (LGT) virus (TP21 strain) is the least virulent of all TBEV-complex flaviviruses. It has not been associated with any human disease. The sequence of wild type virus (TP21 strain) and a more attenuated strain of LGT derived from it (strain E5) were determined. Comparison of these sequences with other TBEV indicated that there is an inverse relationship between the length of the 3' noncoding region and the virulence of TBEV flaviviruses for humans and animals. Analysis of the TP21 and E5 genome sequences revealed only 11 amino acid differences in the polyprotein. We recovered two viable chimeric viruses that contained prM and E genes of LGT virus strain TP21 or E5 and all other sequences from DEN4. These chimeras differed in their LGT-derived sequences at only four amino acid positions in the E protein. In contrast to parental TP21, E5 and DEN4 viruses, chimeric TP21/DEN4 and E5/DEN4 viruses were restricted in growth in primate cells and did not form plaques. Studies in suckling mice inoculated IC with the TP21/DEN4 or E5/DEN4 chimera indicated that these viruses retained the low neurovirulence of their DEN4 parent rather than the higher neurovirulence of their LGT virus parent. Similarly, the LGT/DEN4 chimeric viruses exhibited little if any of the peripheral invasiveness of their LGT parent. A high titer of neutralizing antibodies developed in these mice which were resistant to subsequent peripheral challenge with 1000 IP LD50 of wild type LGT virus. We conclude that (i) the LGT E protein (and possibly the M protein) represents the major protective antigen(s), which induces resistance to lethal LGT challenge, and (ii) proteins encoded by other regions of the LGT genome are required for LGT to spread from a peripheral site to the brain.